GSM7049566: Cell hashing library; Mus musculus; RNA-Seq
Single-cell suspensions from tumors (n = 4 per condition) were prepared as described above except as follows. Cells were purified via an enrichment for CD90.2+ cells using EasySep™ Mouse CD90.2 Positive Selection Kit II (STEMCELL, 18951). Samples were stained using anti-TCRβ (Alexa Fluor® 700, BD Biosciences, 560705), anti-CD4 (BUV395, BD Biosciences, 563790), anti-CD8a (BUV737, BD Biosciences, 612759), and each sample was stained with TotalSeq™-C03NN anti-mouse Hashtag 1 Antibodies with unique hashtag oligonucleotides (HTOs) in staining buffer for 30 min on ice (Biolegend Cat # 155861, 155863, 155865, 155867, 155871, 155873, 155875, 155877). For dead cell exclusion, cells were stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, L34957) for 10 min on ice. Doublets and dead cells were excluded. From each animal, 8,000 CD8+ live cells were sorted using a 5-laser Aria-10 cytometer, and samples from the same condition were pooled in PBS + 0.04% BSA. GEM formation and library preparation was performed using Chromium Next GEM Single Cell 5' v2 workflow from 10x Genomics (Chromium Next GEM Single Cell 5' Kit v2, 16 rxns PN-1000263, Library Construction Kit, 16 rxns PN-1000190, 5' Feature Barcode Kit, 16 rxns PN-1000256, Chromium Next GEM Chip K Single Cell Kit, 48 rxns PN-1000286, Dual Index Kit TT Set A, 96 rxns PN-1000215, Dual Index Kit TN Set A, 96 rxns PN-1000250). Hashtagged cells from 8 mice (4 PBS treated and 4 L. reuteri treated) were pooled onto a single lane of the 10x chip to allow sample multiplexing. Prepared libraries were quality-checked on Agilent TapeStation and sequenced on HiSeq4000 (Novogene Inc).